Beneficial effects of plant volatile oils

ABSTRACT

Plant volatile oils have been found to have certain beneficial effects on the body of a human or non-human. The effects described relate to the maintenance of levels of polyunsaturated fatty acids (PUFAs), the prevention or mitigation of deleterious changes in nervous tissue, elevation of protein levels, and the prevention or mitigation of retinal degeneration.

BACKGROUND OF THE INVENTION

This invention relates to the beneficial effects of plant volatile oilsin a human or non-human body.

Oils from various sources have been known to have a beneficial effect inhumans and animals and can provide vitamins, fats and otherlife-enhancing chemicals. Examples of such oils are fish oil, such ascod-liver oil.

Plant volatile oils, in particular from Thymus vulgaris, have been shownby the inventors to have beneficial effects on various tissues in thebody.

From the present data, it appears that at least some of the beneficialeffects of plant volatile oils on human or non-human bodies areconnected with the levels of polyunsaturated fatty acids (PUFAs) in thebodies.

SUMMARY OF THE INVENTION

According to the present invention there is provided the use of a plantvolatile oil or a constituent thereof

(a) to maintain the levels of PUFAs; and/or

(b) to combat deleterious changes in nervous tissue; and/or

(c) to combat changes in the level of a neuropeptide in nervous tissue;and/or

(d) to produce elevated protein levels; and/or

(e) to combat the effects of ageing by elevating protein levels; and/or

(f) to combat protein loss; and/or

(g) to produce elevated PUFA levels in the retina; and/or

(h) to combat retinal degeneration

in a human or animal body.

DETAILED DESCRIPTION OF THE INVENTION

The term "plant volatile oil" is used herein to refer to any organic oilor fatty substance derivable from plants by distillation and includessynthetic equivalents of such volatile oils-as well as equivalent oilsfrom other, non-plant, sources. A constituent of a plant volatile oil isany ingredient found in a plant volatile oil which causes or contributesto the effect required in the invention.

The term "combat" as used herein refers to the prevention of a condition(ie prophylactic use) as well as treatment of an existing condition toameliorate that condition or to delay or prevent its furtherdeterioration.

The "deleterious changes in nervous tissue" mentioned above in part (b)may comprise degeneration of nervous tissue, changes in morphology orstructure of nerve cells present in the tissue, reduction in the numberof nerve cells present in the tissue, and reduction in nervous tissuefunction (not necessarily accompanied by morphological changes).

In a preferred aspect the present invention provides the use of a plantvolatile oil for the purpose of any of parts (b) to (h) above.

Further according to the invention there is provided the use of a plantvolatile oil or a constituent thereof for the manufacture of amedicament for the purpose of any of parts (a) to (h) above.

In another preferred aspect the present invention provides the use of aplant volatile oil for the manufacture of a medicament for the purposeof any of parts (b) to (h) above.

Further according to the invention there is provided a method of

(a) maintaining the level of PUFAs; and/or

(b) combatting deleterious changes in nervous tissue; and/or

(c) combatting changes in the level of a neuropeptide in nervous tissue;and/or

(d) producing elevated protein levels; and/or

(e) combatting the effects of ageing by elevating protein levels; and/or

(f) combatting protein loss; and/or

(g) producing elevated PUFA levels in the retina; and/or

(h) combatting retinal degeneration

in a human or animal body, said method comprising administering a plantvolatile oil or a constituent thereof to said body.

Preferably, the plant volatile oil is administered in a sufficientconcentration to prevent substantial diminution of the level ofessential long chain PUFAs of metabolic significance, such asarachidonic acid, eicosapentanoic acid and/or docosohexaenoic acid inthe body. The plant volatile oil may be administered in a daily amountof not less than 15 mg per 50 kg of animal body weight, preferably notless than 20 mg per 50 kg and most preferably not less than 25 mg per 50kg.

Plant volatile oils which have been found to have the beneficial effectsstated above are those derivable from clove, nutmeg, pepper, thyme,paprika, oregano, marjoram, basil and French tarragon. Most preferably,the plant volatile oil is derived from thyme or clove. Oils orconstituents thereof may be used alone or in combination with other oilsor constituents.

The neuropeptide mentioned is generally selected from neuropeptide Y,substance P, somatostatin, vasoactive intestinal polypeptide, serotoninand dopamine B-hydroxylase.

The nervous tissue mentioned may be, but is not limited to, smallintestinal neural tissue.

Optionally, the protein levels mentioned in parts (d) and (e) areelevated to the extent that the ratio of creatinine excretion (in mg per24 hours) to 3-methyl histidine excretion (in μmol per 24 hours) isreduced by about a factor of 2-3.

The plant volatile oil or constituent thereof may be administeredtogether with a pharmaceutical carrier and may be simply admixedtherewith or chemically linked thereto. Alternatively the plant volatileoil or constituent thereof may be administered as an emulsion with anaqueous constituent. If required, the plant volatile oil may beencapsulated.

The plant volatile oil or a constituent thereof may be administered byany suitable route, including enteral, parental or topicaladministration.

Maintenance of PUFA levels in the retina has a beneficial effect on theretina, reducing the rate of retinal degeneration (due to age or otherconditions).

"Retinal degeneration" refers to any condition which affects the retinalstructure and/or causes an effect on vision. Mention may be made ofage-related macular degeneration (AMD) as being an important example ofvisual impairment.

Hepatic damage due to intake of free-radical inducing compounds such assolvents and alcohol may be minimised by the administration of plantvolatile oils.

The PUFA status in the foetus/neonate is as important as that in theelderly. Due to the small molecular weight of the oil constituents, itappears likely that they will cross the placenta from mother to foetus,enabling protection from a very early stage of development.

Embodiments of the invention will now be described with reference to thefollowing examples and to the accompanying drawings in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a light microscopic picture of the neuropeptide Yimmunoreactive nerve cell bodies and fibres in the myenteric plexus of acontrol animal (bar scale=100 μm);

FIG. 2 shows a part of a myenteric plexus after treatment with thymeoil, arrows show the large number of neuropeptide Y immunoreactive nervefibres (bar scale=100 μm);

FIG. 3 shows a part of the nerve bundle from aged controls, the arrowpoints to the degenerated nerve fibre among the unstained ones (barscale=1 μm);

FIG. 4 shows part of the inner circular layer after treatment of Thymusvulgaris volatile oil, the arrow shows the somatostatin immunoreactivenerve fibre close to the smooth muscle cell (bar scale=1 μm); and

FIG. 5 shows a part of the tunica mucosa of the small intestine afterthyme oil treatment, the arrow points to the vasoactive intestinalpolypeptide (VIP) immunoreactive nerve fibre in a very close situationto a capillary (bar scale=1 μm);

FIG. 6 shows a graph of the ratio of organ weight to whole body weightin rats following treatment with plant volatile oils (mean±standarddeviation); and

FIG. 7 shows a graph of the ratio of degradation percent per day ofmyofibrillar protein in rats following treatment with plant volatile oil(mean ±standard deviation).

EXAMPLE 1

The use of plant volatile oils to maintain levels of PUFAs.

Preliminary screening of plant volatile oils showed thyme oil and anumber of its constituent compounds to possess strong antioxidativeproperties and thereby to increase levels of PUFAs. The five most activeconstituents were linalool, thujone, camphene, carvacrol and thymol.

Materials and Methods

Test Animals

The studies were performed on groups, 10 in each group, of young (6month) and ageing (22 month) in-bred male CBA/Ca mice (LATI, Godollo,Hungary). The mice were housed in standard plastic cages. The mice werefed a standard laboratory pelleted diet of uniform lipid quantity andquality (LATI, Godollo, Hungary) which, with tap water, was available adlibitum. The treated mice each received for a duration of five weeks(young mice) and 21 weeks (ageing mice) 720 μg of the plant volatileoils administered orally every second day, via a dropper. The dose,following appropriate dilution with drinking water, was emulsified usinga vortex mixer. The doses of volatile oils received by the treated micewere based on formulae worked out from the relative proportions ofintensive growth and the plateau of relatively constant body weightexperienced by the CBA/Ca mice exhibiting a median survival of <24months. The untreated groups of mice received only water. Death was bydecapitation and the livers were immediately removed for analysis.

Volatile Oils

Plant volatile oils were obtained from Serva, Heidelberg, Germany.Volatile oils of almond, clove, nutmeg, pepper and thyme were stored inthe dark at 4° C. until used. The antioxidative capacity was determinedqualitatively by a simple agar diffusion technique of Arujo and Prattusing β-carotene and linoleic acid. Test substances were placed in wells(4 mm in diameter) punched in the agar (Oxoid, Basingstoke, England)followed by, incubation, in the dark, at 45° C. for 4 hours. Subsequentinspection of the plates revealed the extent to which the volatile oilswere antioxidative as measured by the zone of colour retention aroundthe well. Corroborative evidence of the antioxidant capacity wasachieved by using the thiobarbituric acid technique and comparing withbutylate hydroxyanisole (BHA) at the same concentration.

Lipid Analysis

Total lipid was extracted from homogenates of the livers bychloroform:methanol (2:11, v/v) and subsequent aqueous washing accordingto standard procedures. Total phospholipid was separated by thin layerchromatography on silica gel by using a solvent system ofhexane:dimethyl ether:formic acid (80:20:1, v/v/v). Followingvisualisation under UV light, the phospholipid was quantitatively elutedfrom the silica gel with 3×10 ml of chloroform:methanol:water (5:5:1,v/v/v/). Following transesterification by refluxing withmethanol:toluene:sulphuric acid (20:10:1, v/vv/v), the fatty acids weredetermined as their methyl ester derivatives by gas liquidchromatography on a packed column of 15% CP Sil 84 on Chromosorb W(Chrompak, Middleburg, The Netherlands) at 190° C. Quantification of thefatty acid peaks was by electronic integration.

Results

The relative concentrations of the major individual fatty acids withinthe phospholipid fractions of the livers from the young and ageing miceare given in Table 1.1. Treatment with volatile oils had no effect onthe fatty acid composition of the liver phospholipids from the youngmice. Comparison of the fatty acid composition of the phospholipids fromthe young untreated mice with those from the 22 month-old untreated miceshow clearly a large effect of ageing on the relative level ofsaturation/unsaturation within the fatty acids. Total levels of themajor C20 (arachidonic) and C22 (docosohexaenoic) polyunsaturated fattyacids were more than halved being reduced from 22% to only 9% of totalfatty acids. A smaller reduction occurred in the level of linoleic acidwhile there was a compensatory increase in the level of palmitic acid.Dietary administration of the volatile oils to the ageing mice clearlyhad a marked effect on fatty acid distribution by virtually restoringthe proportions of the polyunsaturated fatty acids within thephospholipids to their levels observed in the young mice. Withphospholipids being a predominant component of the total liver lipid,and by far the major carrier of polyunsaturated fatty acids, inparticular the C20 and C22 polyunsaturated fatty acids, the observationstherefore reflected polyunsaturated fatty acid compositional changes inthe liver as a whole.

By using the simple diffusion test for antioxidative properties, anumber of thyme oil constituents have been studied (Table 1.2). Thujone,linalool, camphene, δ-terpinene, β Caryophylene, borneo, myrcene, thymoland carvacrol all had zones of colour retention in excess of 10 mm.

As part of the balance between pro- and antioxidant factors in tissuemetabolism, the levels of polyunsaturated fatty acids play a vital rolein determining their own destiny. From the present observations, itappears that factors present within specific plant volatile oils,notably thyme, can alter significantly this balance in favour of tissueretention of polyunsaturated fatty acids.

Furthermore, in animals fed with plant volatile oils, elevated levels ofthe enzyme glutathione peroxidase (GP) and superoxide dismutase (SOD),key enzymes in the protection against oxidation of lipids, were found.

The antioxidative compounds do not necessarily reside in the volatileoil fraction; for example, the oils from Rosmarinus officinalis andSalvia officinalis do not demonstrate any strong antioxidativeproperties, yet these two plants are among the most-quoted species interms of possessing antioxidants (Table 1.3). A number of solventextractions (hexane, dichloromethane, ethanol and chloroform) were madeon dried plant material and evaluated by the screen described earlier.This revealed a wider range of antioxidant-containing species.

EXAMPLE 2

Effects of plant volatile oils on nervous tissue.

Changes in the number and the distribution of the different nerveelements in old rats were studied after a four month treatment of thymeessential oil using various antisera with the aid of light and electronmicroscopy. In the control (untreated) animals a number of the differentneuropeptide-containing nerve fibres were found in all layers of thesmall intestine. They were frequently observed around the blood vessels,in the inner circular layer and beneath the epithelial lining. Afterthyme oil treatment, the quantity of the immunoreactive nerve fibres wasmore apparent than that of the old controls. The number of theneuropeptide Y and dopamine β-hydroxylase neurolal processes wereincreased in comparison to the untreated old rats.

Materials and Methods

RLEF1/LATI female rats of 26 months of age, weighing 380 g were used.One group of animals was given via dropper orally every second day 2.4mg/100 g bw of volatile oil from Thymus vulgaris L. (Serva, Heidelberg,Germany). For application, the oil was emulsified to an appropriatedilution with drinking water using a vortex mixer before use. The ratsreceived this volatile oil for a period of four months.

The second group of animals, controls, received tap water only. The ratswere fed ad libitum a standard laboratory diet (LATI, G0dollo, Hungary)throughout the experiment and tap water was freely accessible.

At the end of the experimental period, the animals were sacrificed bydecapitation and the tissues immediately isolated and perfused withfixative containing 2% paraformaldehyde, 0.1% glutaraldehyde and 150 mlsaturated picric acid in 0.1M phosphate buffer (pH 7.3). Pieces of thesmall intestine were removed and placed overnight in glutaraldehyde-freefixative at 4° C. Sections, 40 μm thick, were cut with a Vibratome andthe sections rinsed over a period of 24 h in several changes ofphosphate-buffered saline.

Immunostaining was performed according to the peroxidase-antiperoxidase(PAP) technique of Sternberger et al (1970). After peroxidase reaction,the sections were post fixed in osmium tetroxide and embedded inDurkupan ACM (Fluka, Switzerland). The primary antisera were diluted1:500 or 1:5000. Incubation was performed at 4° C. for 48 h. Normalserum and PAP were used in dilutions 1:50. The following antisera wereused: neuropeptide Y (NPY), substance P (SP), somatostatin, vosoactiveintestinal polypeptide (VIP), serotonin and dopamine β-hydroxylase. Allantisera were raised in rabbits. Tissue-bound peroxidase was visualisedwith the diaminobenzidine (DAB) chromogen reaction.

In the controls, complete lack of staining was found followingassessment under conditions of: (1) Omission of the primary antibody;(2) Incubation with primary antibody preabsorbed (12-24 h at 4° C.) withappropriate peptide or other peptides, at a concentration of 10 μM.

Results

In the control (untreated) rat, the NPY immunoreactive nerve elementswere most numerous in all layers, but especially around the bloodvessels (FIG. 1). Thyme oil treatment resulted in an increased quantityof immunoreactive nerve processes compared to the untreated controls(FIG. 2). Under electron microscopic investigation, in the controlanimals the majority of the nerve fibres displayed a general lack ofstaining and in many instances there was an apparent degenerativecondition (FIG. 3). Innervation by SP was associated with a homogeneousdistribution of somatostatin, but the somatostatin immunoreactive nervefibres were more numerous compared to those of SP immunoreactive ones.SP-containing nerve fibres were found in all layers of the smallintestine except the outer longitudinal muscle layer. Small bundles ofSP-containing nerve fibres were visualised running along the submucosalblood vessels. Numerous immunreactive nerve cell bodies were observed inthe myenteric plexus but only a few were found in the submucous plexus.

After thyme oil treatment, nerve fibres either singly, a few together orin small bundles, were observed running along the smooth muscle cells inthe circular muscle layer (FIG. 4). Somatostatin nerve cell bodies werefound to predominate in the submucous plexus. Stained profiles werefound to be located in the core of the villi and in the connectivetissue distributed among the crypts, opposed to epithelial cells, theendothelial cells of blood vessels or smooth muscle cells. VIPimmunoreactive nerve cell bodies were found mainly in the myentericplexus. A large number of VIP immunoreactive nerve fibres were observedaround the blood vessels in the thyme oil treated animals (FIG. 5).Conventional synaptic junctions between the labelled and unlabellednerve cell somata were rarely seen in both plexa. Serotoninimmunoreactive nerve profiles were seldom observed. Dopamineβ-hydroxylase immunoreactive nerve fibres were routinely seen around theblood vessels in all layers of the small intestine.

For all animals, areas of 5000-6500 μm² of intestinal tissue wereexamined and the number of immunoreactive nerve terminals was calculatedfor 1000 4 μm² tissue area. In the thyme oil treated rats, the number ofNPY and dopamine β-hydroxylase immunoreactive neuronal processes wasapparently increased in the experimental animals in comparison with theuntreated rats (Table 2.1).

                  TABLE 2.1    ______________________________________    Influence of a plant essential oil (Thymus vulgaris L)    on the immunoreactive nerve fibres in the small intestine    (number/1000 μm.sup.2 tissue area).    Animal  VIP      NPY    SP      Serotonin                                           DBH    ______________________________________    Control ++++     +++    ++++    +      +    Treated +++      ++++   ++++    +      ++    ______________________________________     The number of fibres identified with the antibodies has been expressed     semiquantitatively from single (+ = 1-5), moderate (++ = 6-10), numerous     (+++ = 11-15) to very dense (++++ = >16 fibres/1000 μm.sup.2 tissue     area).

Discussion

The present data shown that following long term dietary treatment withthyme oil there was an increase in the number of immunoreactive nervefibres within the submucous plexus. As a result, it can be proposed thatdietary intake of thyme oil exerts a beneficial effect on the quantityof the different neuronal elements of the small intestine during ageing.Marked decreases were noted in the activity and amounts of theimmunoreactive nerve elements in all layers of the intestinal walls ofsenile rats.

It has already been shown that free radicals damage the cells mainly byinitiating peroxidation of membrane lipids (Halliwell, 1981). Theimmunoreactive nerve terminals seem to be a target of this process.Indeed, it has been suggested that one of the consequences of freeradical attacks could be the marked change in molecular composition ofthe myelin with ageing (Malone and Szoke, 1992). Most notably this hasbeen observed to include a modified ratio between the unsaturated andsaturated long-chain fatty acids.

The present results show that plant essential oils may be capable ofmitigating these damaging effects.

EXAMPLE 3

The beneficial effect of plant volatile oils on the retina.

Age-related macular degeneration (AMD) is one of the leading causes ofsevere visual impairment in European counties and the United States ofAmerica (Vinding et al, 1992). The primary lesion appears to be in theretinal pigment epithelium (RPE) as the result of a continuousaccumulation of lipofucsin granules during ageing. One possibleexplanation for a predilection to AMD is an increased phagocytotic andmetabolic load on the RPE within the macula giving rise to apreferential accumulation of lipofucsin in these cells and ultimately tophotoreceptor death (Dorey et al, 1989).

The etiology of AMD is at present unknown. Numerous risk factors areknown to be involved that include family history, ocular pigmentation,elastic degeneration of the skin, hyperopia, cardiovascular disease andcigarette smoking (Vinding et al 1992: Blumenkranz, Russell and Robey1985). Nutritional factors may also contribute to AMD. Thus whereas zincis an essential element for normal metabolism its toxic side effectshave been implicated in AMD (Tsao, 1985: Newsome et al 1988).

Materials and Methods

Animals

Eleven month old female LATI rats (Godollo, Hungary) were randomlyassigned into control and 5 treatment groups each of 6 animals. The ratsreceived a standard pelleted diet which with water was available adlibitum. The treated animals each receive via a dropper for a period of17 months an appropriate volume of a water emulsion containing 7.7 mg ofplant volatile oil, administered orally every second day; the controlgroup received water only. The plant oils--almond clove, nutmeg, pepperand thyme (Serva, Heidelberg, Germany)--were stored in the dark at 4° C.until used. Maintenance of their antioxidative capacities was monitoredby the agar diffusion technique described previously. The rats werekilled at 28 months of age and following rapid enucleation, the retinaswere excised and immediately stored in liquid nitrogen to awaitanalysis. To enable sufficient lipid material to be obtained; all theretinas from each treatment group were pooled for analysis.

Lipid analysis

All chemicals used were of the highest specification. Total lipid fromthe retinas was extracted by established procedures involvingchloroform-methanol (Folch, Lees and Stanley, 1957), the lipidultimately being taken up in benzene containing 0.25 percent (W/v)α-tocopherol as an antioxidant. The liquid was then fractionated bystepwise mini-column silicic acid/celite chromatography into 3 discretefractions, total neutral lipid, glycolipid and total phospholipid, byelution with suitable aliquots of chloroform, chloroform:methanol (9:1v/v) and pure methanol respectively. Total phospholipid wastransmethylated by alkaline reduction (Piretti et al 1988) and followingpurification by thin layer chromatography, the fatty acid methyl esterswere quantified by gas chromatography on a fused silica polar capillarycolumn using an appropriate temperature programming and electronicintegration. Confirmation of the identification of the fatty acid methylester was performed by separation as above but in conjunction with massspectrometry.

Results

There was no significant difference in the percentage distribution ofthe eluted retinal lipid fractions between the groups of rats withphospholipid accounting for 24.2±4.1 (S.E) percent of total lipidseparated. Table 3.1 shows the distribution of the unsaturated fattyacids (major acids, percent of total present) associated with thephospholipid fraction of the retinas from the rats following treatment.As can be seen the 3 major unsaturated fatty acids by far in alltreatment groups were oleic, arachidonic and docosahexaenoic acidsaccounting for in excess of 80 percent of the total with docosahexaenoicacid predominating in each case. Treatment with the volatile oils fromclove, nutmeg, pepper and thyme all increase markedly the proportion oftotal polyunsaturated fatty acid, accounted for in each case by verysubstantial increases in the levels of both arachidonic anddocosahexaenoic acids with concomitant large decreases in the levels ofpalmitoleic and oleic acids. There was also a decrease in the level oflinoleic acid.

Polyunsaturated fatty acids comprised by far the major proportion of thetotal unsaturated content within the retinal phospholipids or the 28month rats with docosahexaenoic acid predominating. The administrationdaily of 3.4 mg of the volatile oils from clove, nutmeg, pepper andthyme over a period of 17 months resulted in the maintenance of verymuch higher levels of polyunsaturated fatty acids, in particulardocosahexaenoic acid, within the retinal phospholipids. This increaseoccurred mainly at the expense of a reduction in the level of oleicacid.

The present observations sustain the suggestion of the involvement of anantioxidant role in retinal function during ageing. The essential oilsof clove, nutmeg, pepper and thyme all afforded considerable protectionin the maintenance of polyunsaturated fatty acid levels, in particularthe highly labile docosahexaenoic acid.

As the n-3 polyunsaturated fatty acids, in particular docosahexaenoicacid, are essential for the normal electrical response in visualexcitation (Neuringer, Anderson and Connor, 1988) a decrease in theirconcentrations must play an important role amongst the causes of visualimpairment which accompany senescence. There is no proven treatment forAMD except laser photocoagulation in choroidal neovascularisation. Thepresent invention is therefore of significance from both clinical andtherapeutic points of view.

EXAMPLE 4

The beneficial effects of plant volatile oils on protein levels.

Groups of rats were dietarily administered regular small doses of aselection of plant volatile oils between 28 and 45 months of age.Treatment with the oils resulted in an enhancement of whole body weightand overall reductions in specific organ to whole body weight. Urinaryexcretion of 3-methyl-histidine was reduced in all cases butparticularly in those rats that received the volatile oils from cloveand thyme. Myofibrillar protein breakdown was also most reduced by theoils from clove and thyme.

Measurements of the overall rates of protein synthesis and degradationin muscle are able to establish existing states of protein metabolism. Awide variety of physiological and pathological conditions have beenidentified under which muscle becomes a subject of net protein breakdowndue to the rate or protein degradation exceeding thank of synthesis(Tischler, 1981).

Materials and Methods

Animals

Female RLEF1/LATI rats (Godollo, Hungary), 28 months of age, were thesubject of the experiments. The rats were matched for weight and eachreceived ad libitum a standard laboratory compounded diet with a proteincontent of 20.2 percent; tap water was freely available. The rats wererandomised into control and experimental groups, the latter to receivelong term dietary treatment with a single plant volatile oil. The oilswere bitter almond, clove, nutmeg, pepper and thyme (Serva, Heidelberg,Germany). In each case the oil was emulsified with water and given 3times per week via a dropper at a rate of 7.7 mg for a period of 17months. At the completion of the dietary period the rats were killed bydecapitation and the major organs excised for analysis.

Analysis

Protein metabolism was monitored through comparative outputs of3-methyl-histidine and creatinine as detailed previously (Albrecht etal, 1992). Urine samples were prepared for the quantification of3-methyl-histidine by precipitation of the protein with perchloric acidand following washing, neutralisation with potassium hydroxide,lyophilisation; hydrolysis with 6N hydrochloric acid and finallyevaporation to dryness. Samples were stored frozen in sodium citratebuffer, pH 2.2 prior to analysis. Norleucine was used as an internalstandard, 3-methyl-histidine was separated by means of ion exchange(Aminex A5, Bio-Rad, Germany) high performance liquid chromatography(Waters millipore, Germany) with post column ninhydrin derivatisation(Spackman et al, 1958). The eluate was channelled into an automaticanalyser (ADM 300, Medingen, Germany) for the detection of ninhydrinpositive compounds. Creatinine was quantified by a modified Jaffeprocedure.

Statistical differences between means were investigated by the Students't-test and those among more than 2 means by applying the Newman-Keulsmultiple range test when the analysis of variance gave a significant(P<0.05)F-value.

Results

Table 4.1 shows the whole body and individual organ weights of the ratsfollowing treatment for the period of 17 months. As can be seen, theadministration of the various plant oils considerably enhanced wholebody weight compared to untreated controls; the increases ranged from 22percent in the case of the rats that received treatment with thevolatile oil extract from pepper to 43 percent in the rats that receivedthe volatile oil from nutmeg.

As exemplified by the liver and spleen (FIG. 6), treatment with thevolatile oils reduced overall the ratio of specific organ weight towhole body weight.

Table 4.2 shows the measurements for the rates of excretion of3-methyl-histidine both in absolute amounts per unit period of time andper unit of body weight and standardised relative to the excretion rateof creatinine. It is clear that in terms of body mass, treatment withthe volatile oils other than that of nutmeg caused a reduction in theexcretion of 3-methyl-histidine. When related to that of creatinine, theexcretion of 3-methyl-histidine was reduced in all cases of oiladministration but in particular as a result of treatment with that fromclove and thyme for which the reductions in excretion were significant,P<0.05 and P<0.01 respectively.

FIG. 7 shows the values obtained for the fractional rates of degradationof myofibrillar protein as a result of the treatment with the volatileoils. As in the case of overall excretion, treatment with the volatileoils other than that of nutmeg reduced myofibrillar protein breakdownwith the largest effect occurring as a result of treatment with oilsfrom clove and thyme.

The present results clearly indicate that the dietary administration ofplant volatile oils, in particular those from clove and thyme, display asparing effect on the metabolism of senile rats in favour of body massretention.

A combination of urinary creatinine and 3-methyl-histidinequantifications is able to provide an accurate index of muscle proteinturnover, the creatinine measurements providing a reliable indicator ofthe quantity of muscle protein mass (Forbes and Bruining, 1976) and3-methyl-histidine, because of its specific association with the muscle,an indicator of protein breakdown (Yong and Munro, 1978). Indeed thedecline in muscle mass based on creatinine output with age has beenshown to be similar to the decrease in the rate of muscle proteinturnover based on urinal 3methyl-histidine excretion (Prothro, 1989).

In quantitative terms the level of 3-methyl-histidine excretion isdependent upon the combination of the rate of muscle catabolism andmuscle pool size, although circumstances have been demonstrated underwhich increased protein turnover occurred but which was not associatedwith increased nitrogen loss (Wilson et al, 1981). In rats, as in arange of other animal species, it has been established that increasingage in accompanied by an overall decrease in the rate of myofibrillarprotein degradation (Santidrian et al, 1981). It is clear from thepresent measurements of 3methyl-histidine outputs that administration ofplant volatile oils reduced significantly muscle protein breakdown.

Modifications and improvements may be incorporated without departingfrom the scope of the invention.

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                                      TABLE 1.1    __________________________________________________________________________    The fatty acid compositions (major acids weight percentage of total) of    the phospholipid fraction    of the livers from the young (6 months old) and ageing (22 months old)    CBA/Ca mice.                        Ageing Mice            Young Mice        Treated    Fatty Acid            Untreated                  Treated                        Untreated                              Clove Thyme Nutmeg                                                Pepper                                                      Almond    __________________________________________________________________________    Palmitic            26.2 ± 0.54                  25.3 ± 0.89                        38.3 ± 0.34.sup.3                              35.3 ± 1.00.sup.a                                    31.6 ± 2.49.sup.a                                          31.5 ± 1.28.sup.c                                                29.2 ± 2.44.sup.b                                                      35.1 ± 2.55.sup.    Palmitoleic            1.98 ± 0.14                  1.87 ± 0.05                        2.70 ± 0.16.sup.2                              2.02 ± 0.10.sup.b                                    1.54 ± 0.33.sup.a                                          2.19 ± 0.13.sup.a                                                1.85 ± 0.11.sup.b                                                      2.54 ± 0.13.sup.    Stearic 15.7 ± 0.69                  14.6 ± 0.33                        16.4 ± 0.44.sup.                              14.8 ± 0.53.sup.                                    16.2 ± 1.74.sup.                                          13.5 ± 1.04.sup.a                                                14.2 ± 0.51.sup.a                                                      16.0 ± 0.87.sup.    Oleic   14.9 ± 0.77                  13.6 ± 0.47                        17.9 ± 1.48.sup.                              16.0 ± 1.34.sup.                                    13.3 ± 1.13.sup.a                                          16.2 ± 1.03.sup.                                                15.5 ± 0.81.sup.                                                      15.6 ± 0.49.sup.    Linoleic            18.6 ± 1.00                  16.9 ± 0.25                        14.1 ± 0.52.sup.2                              15.8 ± 0.61.sup.                                    16.5 ± 0.78.sup.a                                          15.5 ± 1.07.sup.                                                18.2 ± 0.18.sup.                                                      16.7 ± 0.82.sup.a    Linolenic            0.60 ± 0.15                  0.42 ± 0.03                        0.70 ± 0.04.sup.                              0.64 ± 0.09.sup.                                    0.35 ± 0.06.sup.b                                          0.33 ± 0.06.sup.c                                                0.42 ± 0.03.sup.c                                                      0.39 ± 0.09.sup.a    Eicosatrienoic            2.04 ± 0.25                  2.32 ± 0.11                        0.76 ± 0.10.sup.3                              1.42 ± 0.36.sup.                                    1.08 ± 0.19.sup.                                          1.13 ± 0.12.sup.a                                                1.62 ± 0.25.sup.c                                                      1.30 ± 0.17.sup.a    Arachidonic            15.5 ± 0.91                  16.5 ± 0.98                        6.60 ± 0.44.sup.3                              10.3 ± 0.98.sup.b                                    14.5 ± 1.89.sup.b                                          14.0 ± 0.74.sup.c                                                14.0 ± 1.09.sup.c                                                      9.12 ± 7.46.sup.    Docosapentaenoic            0.18 ± 0.07                  0.28 ± 0.03                        <0.10 0.31 ± 0.08.sup.                                    <0.10 0.17 ± 0.05.sup.                                                0.19 ± 0.07.sup.                                                      0.10    Docosahexaenoic            3.86 ± 0.35                  5.87 ± 0.64                        1.68 ± 0.32.sup.3                              3.31 ± 0.32.sup.b                                    4.33 ± 1.00.sup.a                                          5.61 ± 0.64.sup.c                                                5.34 ± 0.58.sup.c                                                      3.31 ± 0.70.sup.    __________________________________________________________________________                                                      1     Each result is the mean ± standard error of 10 observations.     Significance of difference, using Student's test, from young control mice     .sup.1 = P < 0.05; .sup.2 = P < 0.01; .sup.3 = P < 0.001     Significance of difference from aged Control mice: .sup.a = P < 0.05;     .sup.b = P < 0.01; .sup.c = P < 0.001

                  TABLE 1.2    ______________________________________    Antioxidative properties of the volatile oil of    Thymus vulgaris (thyme) and its main constituents.    Oil Constituent                 Zone of Colour Retention                                Intensity    ______________________________________    Whole oil    25.0           +++    Borneol      11.4           +    Camphene     16.2           ++    Carvacrol    13.2           +++    β-Caryophyllene                 12.3           ++    1,8-Cineole  Neg            Neg    p-Cymene     Neg            Neg    Linalool     20.6           ++    Myrcene      10.8           ++    Oct-1-en-3-ol                 Neg            Neg    α-Pinene                 Neg            Neg    β-Pinene                 Neg            Neg    α-Terpinene                 Neg            Neg    δ-Terpinene                 12.3           ++    Terpinen-4-ol                 Neg            Neg    α-Thujone                 Neg            Neg    β-Thujone                 19.3           ++    Thymol       13.2           +++    ______________________________________     Diameter of zone of colour retention measured in mm:     colour intensity evaluated by + moderate, ++ average, +++ high, Neg     Negative result.

                                      TABLE 1.3    __________________________________________________________________________    Plant essential oils exhibiting antioxidative and pro-oxidative    properties.    __________________________________________________________________________    Almond Bitter           + Bay    + Caraway                            0 Clove + Fennel Sweet                                             0    Almond Sweet           0 Bergamot                    - Cardamon                            - Coriander                                    - Geranium                                             0    Angelica           0 Calmus + Celery                            + Dill  0 Ginger 0    Anise  0 Chamomile                    0 Cinnamon                            + Estragon                                    - Laurel +    Basil  0 Cananga                    0 Citronella                            + Eucalyptus                                    - Lavender                                             0    Lemon  0 Melissa                    0 Parsley                            + Rosemary                                    + Star Anise                                             0    Lime   - Mint   + Pepper                            + Sage  - Thuja  -    Lovage + Nutmeg + Peppermint                            + St. J Wort                                    0 Thyme  +    Mandarin           0 Orange 0 Pimento                            + Sassafras                                    + Valerian                                             -    Marjoram           0 Orange Bitter                    0 Rose  + Spike + Verbena                                             -    __________________________________________________________________________     + Antioxidant activity;     - Prooxidative activity;     0 No activity

                  TABLE 3.1    ______________________________________    Percentage composition of the unsaturated fatty acid methyl esters    in the retinas from rats fed the essential oils. Each result is    for the pooled samples of the retinas from 6 rats per group.    Treatment Control Almond  Clove                                   Nutmeg                                         Pepper                                               Thyme    ______________________________________    palmitoleic              6.55    3.09    1.96 1.72  1.94  3.28    oleic     30.14   35.02   22.18                                   22.76 23.38 25.00    cis-vaccenic              3.69    3.59    3.98 4.38  4.69  5.01    linoleic  7.02    3.79    2.32 2.64  3.08  2.78    arachidonic              12.41   13.49   15.98                                   15.98 18.50 17.51    eicosamonoenoic              1.00    0.99    0.37 0.89  <0.10 <0.10    eicosapentaenoic              <0.10   <0.10   0.57 <0.10 0.67  0.67    docosahexaenoic              39.18   40.03   52.63                                   51.63 47.73 45.75    ______________________________________

                                      TABLE 4.1    __________________________________________________________________________    Body and organ weights of rats at 28 months.                                              Kidney                                                    Kidney    Body weight              Brain Lung  Heart  Spleen                                       Liver  (left)                                                    (right)    __________________________________________________________________________    Control        239 ± 39.6              1.38 ± 0.03                    4.37 ± 1.01                          1.28 ± 0.05                                 0.42 ± 0.08                                       9.77 ± 1.52                                              1.15 ± 0.08                                                    1.17 ± 0.07    Almond        293 ± 25.5              1.38 ± 0.03                    4.39 ± 0.72                          1.15 ± 0.07                                 0.32 ± 0.04                                       8.03 ± 1.13                                              1.09 ± 0.10                                                    1.08 ± 0.11    Clove        311 ± 20.2              1.39 ± 0.02                    4.20 ± 0.74                          1.20 ± 0.11                                 0.46 ± 0.03                                       10.46 ± 1.30                                              1.21 ± 0.02                                                    1.26 ± 0.12    Nutmeg        340 ± 30.7              1.43 ± 0.04                    5.32 ± 1.15                          1.33 ± 0.18                                 0.34 ± 0.04                                       10.17 ± 1.04                                              1.29 ± 0.07                                                    1.31 ± 0.05    Pepper        290 ± 32.9              1.36 ± 0.06                    4.32 ± 0.56                          1.05* ± 0.06                                 0.29 ± 0.07                                       9.19 ± 1.54                                              1.22 ± 0.10                                                    1.20 ± 0.12    Thyme        298 ± 40.5              1.33 ± 0.05                    4.27 ± 0.54                          1.01 ± 0.14                                 0.30 ± 0.04                                       7.08 ± 0.50                                              1.15 ± 0.09                                                    1.15 ± 0.08    __________________________________________________________________________     Each result is the mean ± standard error.     Significantly different from control * = P < 0.05.

                  TABLE 4.2    ______________________________________    Absolute and relative urinary excretion ratios of 3-methyl-histidine.                       Creatine   Ratio    3 methyl-histidine excretion                       excretion  3-methyl-    μmol per  μmol per kg                           mg per     histidine/    24 h         body weight                           24 h       creatine    ______________________________________    Control           3.08 ± 0.61                     13.1 ± 2.86                               7.72 ± 2.47                                        0.43 ± 0.07    Almond 2.86 ± 8.14 ± 9.83 ±                                        0.29 ±    Clove  2.50 ± 0.40                     7.93 ± 0.87                               10.9 ± 2.70                                         0.23 ± 0.03*    Nutmeg 3.62 ± 0.42                     11.8 ± 1.09                                14.9 ± 0.75*                                        0.24 ± 0.03    Pepper 3.34 ± 0.17                     15.0 ± 2.86                               9.86 ± 0.47                                        0.34 ± 0.01    Thyme  2.64 ± 0.16                     9.33 ± 0.98                                18.5 ± 1.72*                                        0.15* ± 0.01    ______________________________________     Each result is the mean ± standard error.     *Significantly different from control P < 0.05;     ** significantly different from control P < 0.01.

We claim:
 1. A method of producing elevated PUFA levels in nervoustissue in a human or animal body, said method comprising administering aplant volatile oil or an active constituent thereof to said body in aphysiologically effective amount.
 2. A method as claimed in claim 1,wherein the plant volatile oil or constituent thereof comprises oilselected from the group consisting of clove, nutmeg, pepper, thyme,paprika, oregano, marjoram, basil and French tarragon or constituents ofsuch oils, or any mixture thereof.
 3. A method as claimed in claim 1,wherein the PUFAs are n-3 PUFAs.
 4. A method as claimed in claim 1,wherein the PUFAs comprise docosahexaenoic acid.
 5. A method as claimedin claim 1, wherein the plant volatile oil or constituent thereof iscombined with a pharmaceutical carrier or excipient.
 6. A method asclaimed in claim 1, wherein the plant volatile oil or constituentthereof is administered as an emulsion with an aqueous constituent.
 7. Amethod as claimed in claim 1, wherein the plant volatile oil orconstituent thereof is encapsulated.
 8. A method as claimed in claim 1,wherein the plant volatile oil or constituent thereof is administeredenterally, parenterally or topically.
 9. A method of treating nervoustissue in a human or animal body, said method comprising administering aplant volatile oil or an active constituent thereof to said body, in anamount sufficient to elevate the PUFA levels in the nervous tissue. 10.A method as claimed in claim 9, wherein the plant volatile oil orconstituent thereof is administered to the human or animal body in adaily amount of not less than 15 mg per 50 kg of body weight.
 11. Amethod as claimed in claim 9, wherein the plant volatile oil orconstituent thereof comprises oil selected from the group consisting ofclove, nutmeg, pepper, thyme, paprika, oregano, marjoram, basil andFrench tarragon or constituents of such oils, or any mixture thereof.12. A method as claimed in claim 9, wherein the PUFAs are n-3 PUFAs. 13.A method as claimed in claim 9, wherein the PUFAs comprisedocosaexaenoic acid.
 14. A method as claimed in claim 9, wherein theplant volatile oil or constituent thereof is combined with apharmaceutical carrier or excipient.
 15. A method as claimed in claim 9,wherein the plant volatile oil or constituent thereof is administered asan emulsion with an aqueous constituent.
 16. A method as claimed inclaim 9, wherein the plant volatile oil or constituent thereof isencapsulated.
 17. A method as claimed in claim 9, wherein the plantvolatile oil or constituent thereof is administered enterally,parenterally or topically.
 18. A method as claimed in claim 9, whereinthe nervous tissue is retinal tissue.
 19. A method as claimed in claim1, wherein the nervous tissue is retinal tissue.